Amphibia: Gymnophiona), and a Comparison to Dermal Ossifications of Other Vertebrates

Home , Caecilian, Dermophis, Microcaecilia, Microcaecilia unicolor

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JOURNAL OF MORPHOLOGY 2062-43 (1990)

Structure of the Scales of Dermophis and Microcaecika (Amphibia: Gymnophiona), and a Comparison to Dermal Ossifications of Other Vertebrates

LOUISE ZYLBERBERG AND MARVALEE H. WAKE CNRS UA 161 and Laboratoire d’Anatomie Comparhe, Universith de Paris VII, Paris 75251, France (L.Z.);Department of Integrative Biology and Museum of VertebrateZoology, University of California,Berkeley, California 94720

ABSTRACT The structures of the dermal scales and the cells surrounding the scales in two species of gymnophione amphibians were studied using histochemistry and light, scanning and transmission electron microscopy. Scales are composed of a basal plate of several layers of unmineralized collagenous fibers topped with mineralized squamulae. Squamulae are composed of numerous mineralized globules and mineralized, thick collagen fibers. Mineralization is therefore both spheritic and inotropic. Isolated flattened cells lie on the outer surface of the squamulae and seem to be involved in mineral deposition. Cells that line the basal plate synthesize the collagenous stroma of the plate. Each scale lies in a thin connective tissue pocket, and a large connective tissue pouch includes several scales in each annulus. The similarities of gymnophione scales to elasmoid scales of osteichthyans are largely superficial. Aspects of mineralization and of pocket development differ considerably. There are also similarities, as well as differences, in the gymnophione scales and osteoderms of amphibians and of reptiles. We consider that such dermal structures have arisen many times in diverse lineages of vertebrates, and that these are expressions of properties of dermal collagen to support mineralization by specialized dermal cells. However, we recommend that the term “dermal scale” be used for the mineralized dermal units of osteichthyans and gymnophiones, and “osteoderm” for the dermal structures of frogs and squamates, with the understanding that the terminology recognizes certain convergent attributes of shape and structure, but not of process.

The presence of dermal scales that are com- convergent evolution, in response to the struc- posed of an unmineralized base plate of collage- tural constraints of the tissue involved. nous fibers and a superficial layer of mineralized There have been several descriptions of the squamulae is unique to members of the amphib- scales of gymnophione taxa in the last 150 years. ian Order Gymnophiona among all living tetra- Mayer (1829a,b), Rathke (1852), and Leydig pods. Because gymnophione scales are structur- (1853) commented briefly on scale morphology. ally similar to those of teleost fishes, they have Sarasin and Sarasin (1887-90) described the been assumed to be homologous (Zylberberg et scales of Ichthyophis glutinosus from Sri Lanka, al. ’80; Ruibal and Shoemaker, ’84). However, and several subsequent analyses have corrobo- recent examinationof new data on gymnophione rated their conclusions about the relationship of scales, together with an assessment of the mor- the scales to other dermal and to epidermal phology of fish scales, the dermal ossificationsof structures. Cockerell (’11, ’12),Phisalix (’10, ’12), frogs, and the osteoderms of lizards, causes us to Datz (’23), Ochotorena (’32), Marcus (’34),Gabe question the assumption of homology of dermal (’71a,b), Casey and Lawson (’79), Zylberberg et scales. This analysis indicates that all dermal al. (’80), Perret (’82), and Fox (’83)described the ossificationsshare a number of features that are scales of diverse species, usually Ichthyophis or structural properties of the particular organiza- Hypogeophis, at various levels of resolution. Tay- tion of the dermis, and that the similarities and lor (’72) assembled an “atlas of scales” composed differences in scale and osteoderm structure of photographs and descriptions of single scales found in various lineages constitute evidence for from virtually all of the species of gymno- o 1990 WILEY-LISS, INC. 26 L. ZYLBERBERG AND M.H. WAKE phiones. He indicated that features such as scale and Microcaecilia unicolor from Guyana were size, shape, numbers and distribution, and pat- processed for analysis by several techniques. The tern of mineralization might be useful for system- specimens of Dermophis mexicanus from which atic analysis. However, Wake and Nygren ('87) scales were taken are deposited in the collections demonstrated marked individual, ontogenetic, of the Museum of Vertebrate Zoology, Univer- and sexual variation in nearly all scale parame- sity of California, Berkeley, and the M. unicolor ters for Dermophis mexicanus from a single in the Museum National d'Histoire naturelle, population, and suggested that scales are of little Paris. Scales were stripped from the annuli of use as taxonomic characters to diagnose gymno- several formalin-ked, alcohol-preserved speci- phione species. Although Feuer ('62) proposed mens of different sizes (and presumably ages) that scales might provide information about ages and sexes; all scales per annulus were stained of individuals, Zylberberg et al. ('80) presented with alizarin red-S (see Wake and Nygren, '87, evidence to the contrary. for details). Quadrants of skin including several The greatest numbers of scales occur in taxa annuli were fixed in neutral buffered formalin or of gymnophiones considered primitive based on in Bouin's mixture, embedded in paraffin, and other characters (osteology, myology, reproduc- sectioned sagittally or frontally. Stains and his- tive biology; Ichthyophis, Caudacaecilia, Rhina- tochemical reactions are summarized in Tables 1 trerna, Epicrionops-see Taylor, '68, '72; Nuss- and 2. Scales were prepared for scanning elec- baum and Wilkinson, '89). Scales are embedded tron microscopy by excising, mounting on stubs, in virtually all of the annuli (i.e., body rings) of critical-point drying, and sputter-coating with primitive species, and these taxa have several gold or gold-palladium alloy. Scales were exam- annuli per body segment. In gymnophiones, there ined and photographed with an ISI-DS-130dual- is a trend toward the reduction of numbers of stage scanning electron microscope or with a annuli and, concomitantly,of scales. Taxa consid- JEOL 8s M 35 SEM at an operating voltage of ered highly derived based on other characters 25 kV. (e.g., typhlonectids and scolecomorphids)charac- For transmission electron microscopy, small teristically lack secondary annuli and scales (though Wake, ['75] and Moodie, ['78] found pieces of skin were removed from several regions scales in some Typhlonectes). of the body, fixed in a mixture of 2.5 % glutaral- However, the scales of few taxa have been dehyde and 2 % paraformaldehyde in 0.1 M ca- examined in detail with comparison to out- codylate buffer (pH 7.4) for 2 or 3 hr at room groups in mind. We present new data on the temperature; specimens were washed in 0.1 M morphology of scales of Dermophis mexicanus cacodylate buffer containing 10% sucrose, and and Microcaecilia unicolor (both Gymnophiona: postfixed in 1%osmium tetroxide in 0.1 M ca- Caeciliaidae wde Nussbaum and Wilkinson, '891) codylate buffer. Some samples were decalcified to augment the detailed data available for Ich- in the fixative with 0.1 M ethylene-diamino-tetra- thyophis kohtaoensis (Gabe, '71a,b; Zylberberg acetic acid (EDTA) added for 1or 2 days at 4"C, et al., '80; Fox, '83) and Hypogeophis rostratus and then washed and postfixed. Ruthenium red (Casey and Lawson, '79; Zylberberg et al., '80). (Martino et al., '79) was used as an electron The morphology of the scales of D. mexicanus microscopic stain for extracellular polyanions and M. unicolor is compared with that of those (Luft, '71a,b) such as acid mucosubstances and scales described previously, and with that of acid phospholipids. Ruthenium red was added dermal ossifications in other vertebrates. Our to the fixativecontaining EDTA, the wash buffer, comparative morphological approach elucidates and the osmium tetroxide solution. All the fixed the similarities and differences in mineralization samples were dehydrated in ethanol and cleared patterns of such dermal derivatives as scales and in 1-2 epoxy-propane. They were left in a 1:l osteoderms and allows assessment of homology epoxy-propanernpon mixture overnight or versus convergence of teleost scales, caecilian longer, and then transferred to fresh resin. Poly- scales, and anuran and reptilian osteoderms. The merization was carried out at 60°C. Thick sec- study also provides a basis for continued re- tions (-1 pm) were stained with toluidin blue for search into the questions of development and light microscopy. Thin sections of selected areas homologies of dermal structures in vertebrates. were cut with a Reichert ultramicrotome using a diamond knife with the tissue block oriented obliquely to the knife to improve sectioning of MATERIALS AND METHODS hard fibrous tissues (AUizard and Zylberberg, Scales of Dermophis mexicanus from San '82). Sections were mounted on collodion-coated Marcos Prov., Guatemala, and Chiapas, Mexico, grids and stained with uranyl acetate and lead GYMNOPHIONE SCALE MORPHOLOGY 27

TABLE 1. Histologicalstains employed for analysis of scales Histological stain Reference Azan Heidenhain in Gabe ('68) Mallory's azan Humason ('79) Hematoxylin & eosin Humason ('79) One-step trichrome Gabe and Martoja in Gabe ('68) Histochemical reactions Specificity Reference Proteins Danielli's coupled tetrazolium reaction General reaction Gabe ('68) Ferric ferricyanidereaction Reducing groups Adams in Gabe ('68) Carbohydrates Periodic acid-Sch8(PAS) Neutral mucosubstances MacManus in Gabe ('68) Alcian blue pH 2.5 (AB 2.5) Acid mucosubstances Mowry in Gabe ('68) Alcian blue pH 0.5 (AB 0.5) Sulfated mucosubstances Mowry in Gabe ('68) AB2.5 + PAS Neutral and acid muccaubstances Mowry in Gabe ('68) AB 0.5 + PAS Neutral and sulfated mucosubstances Mowry in Gabe ('68) Toluidine blue pH 4.2 Neutral vs. acid (carboxyl-rich and sulfated mucosubstances) Lison in Gabe ('68) Paraldehyde-fuchsian blue pH 2.5 Sulfated mucosubstances Gabe ('68) Danielli's reaction +AB 2.5 Protein and mucosubstances Lillie and Turner in Gabe ('68) Calcium Alizarin red3 Calcium Wake and Nygren ('87)

TABLE 2. Histological and histochemicalcharacteristics of scales of Dermophis mexicanus and Microcaecilia unicolor Scale Basal plate Squamulae Reaction Inner Outer Dermis Azan Red Blue Blue Blue Mallory's azan Blue Red Red Blue Hematoxylin & eosin Pink Purple Purple Pink One-step trichrome Red Green Green Green Picro-ponceau Yellow-pink Dark yellow Red Pink Danielli's reaction ++ +++ +- ++ Ferric ferricyanide - - - - PAS +- +++ ++ + AB 2.5 - ++ ++ - AB 0.5 - + +++ - Toluidine blue Blue Blue or purplish-blue Purplish-blue Blue PAS + AB 2.5 Red Purplish-blue Blue Red PAS + AB 0.5 Red Purplish-red Purplish-blue Red Paraldehyde-fuchsin + PAS Red Reddish-purple Purple Red Danielli's reaction + AB 2.5 Purple Purple and Blue Blue Purple Alizarin red-S - Red Red - citrate (Reynolds,'63). The grids were examined orly in both primary and secondary annuli to in a Philips EM 300 electron microscope at an mid-body, from which point numbers are rela- operating voltage of 80 kV with a cooled anticon- tively constant, although sizes within each annu- tamination device. lus show much variation, until they diminish in the most posterior annuli (see Wake and Ny- RESULTS gren, '87, for data). In both taxa examined, the General organization of the scales scale rows are covered by a thin layer of epidermis (Figs. la, 2), hence are not exposed to the The most anterior scales in Dermophis mexi- environment. Scales are somewhat irregular in canus are located in the tenth to twentieth pri- size and shape, and a diversity of scale sizes and mary annulus (counting posteriorly from the shapes is found in each scale row (Wake and head); their sizes and numbers increase posteri- Nygren, '87). Excised scales treated with alizarin 28 L. ZYLBERBERG AND M.H. WAKE red-S have red-stained structures atop an un- Squamulae are flattest centrally; points and stained base plate, indicating specificity for cal- ridges of more peripheral squamulae are higher cium in the mineralized squamulae; the latter (Figs. 9, 10). At moderate magnification also have been called denticles. We prefer the (- x 1,OOO), they show numerous ridges and folds more precise term squamulae, to avoid possible (Figs. 11, 12). At higher magnification (Fig. 13), confusion of these structures with the “denticles” it is clear that each squamula is an array of of sharks, for example. round, mineralized deposits of varying heights. In parasagittal sections of skin with several Some of the deposits are fused superficially or annuli, the scale pouches are embedded in each linearly; however, the bases of the mineralized annulus and form a ring that partly or com- structures rarely are fused. pletely encirclesthe body. Characteristically, the The points and ridges that form the upper- distal end of the scale pouch lies in the dermis most surface of each squamula include a loose below the epidermis (Figs. la, 2, 3). The distal framework composed of acid mucosubstances margin of the pouch may be deep to small der- that stain with Alcian blue (Fig. 14), whereas mal mucous glands; the proximal end of the abundant neutral mucosubstances and proteins pouch is broader and located at the boundary are located more centrally within the squamula between the superficial and the deep dense der- (Figs. 14,15;Table 2). The uppermost surface of mis that overlies the body wall musculature (Figs. a squamula is composed of numerous mineral- la, 2). Each scale pouch usually is associated ized globules that appear as isolated structures, with large mixed granular (so-called “poison”) particularly at the periphery of the squamula glands both dorsally and ventrally. The pouch is (Figs. 7,24,25), or that aggregate to form large a thin, connective tissue structure (Figs. 2, 3). concretions in the central part of the squamula Each scale lies in its own pocket or sac within the (Figs. 13, 16). The mineral deposit is composed pouch (Figs. 2, p6). Fibroblasts line the inner of crystals that have a radial arrangement within surface of each pocket (Figs. 7, 8), and, within the globules (Figs. 18,22). In demineralized sec- each pocket, the fibroblasts (scleroblasts) that tions, organic matrix cannot be distinguished in form the scale are arranged in layers surround- many globules. They appear as electron-lucent ing each scale. The scleroblasts constitute an circular spaces (Fig. 17). In other globules, a thin uninterrupted layer on the bad surface of the fibrillar network forms a central core surrounded scale. At the scale margin, the collagenous stroma by an electron-lucent space (Fig. 19).An electron- of the scale aligns with the connective tissue of dense opaque material that displays high con- the scale pocket (Figs. Ib, 25). Numerous scales, trast with ruthenium red lines the surface of the contained in their pockets, overlap within a pouch globules (Fig. 19).The central part of the squam- (Fig. 1, 2). All scales lie similarly in the pouch ula contains both mineralized globules and min- with the denticulate surface facing outward. eralized, thick collagen fibrils that arise from the Some large scales are folded back on themselves basal plate (Figs. 16, 23). The crystals are ori- in the scale pockets (Fig. 4). Scale structure ented along the collagen fibrils, and invade the varies with size of scale and with region on the fibrils somewhat. At the inner surface of the scale (central vs. peripheral). Large scales have a squamulae, mineralized globules are inserted in thick base plate composed of as many as seven the network formed by the collagenous stroma layers, or plies, of fibers. The orientation of the (Fig. 23). fibers alternates regularly (Figs. IC,5,13). Miner- Isolated, flattened cells are located on the outer alized squamulae lie atop, and slightly embed- surface of the squamulae (Fig. 13). They contain ded in, the upper layer of the base plate (Figs. IC, a voluminous central nucleus, and are more nu- 3, 13). Each squamula is a discrete structure merous in young animals than in older ones. In (Figs. 3,13). young animals, the cells have long cytoplasmic processes that cover the surfaces of the squamulae, and the processes are near the outer mineral- The squamulae ized globules (Fig. 13). A fuzzy organic material At low magnification, central squamulae on is located near the plasma membrane facing the the base plate appear to be square to round squamula where the first crystal deposits appear shaped, whereas more peripheral squamulae are (Fig. 20), suggesting that these cells are involved more elongate. Squamulae are arrayed in irregu- in the production of the mineralized globules. larly concentric circles (Figs.9,lO) and are highly The crystals first deposited in these globules irregular in shape. The longitudinal sectional have no apparent orientation (Fig. 20). The ra- profiles of the squamulae vary; they are rela- dial organization of the crystals becomes appar- tively flat but bear points and ridges (Figs. 2-5). ent in globules still in the vicinity of the sclero- GYMNOPHIONE SCALE MORPHOLOGY 29

Fig. 1. Diagrammaticrepresentation of a caecilian scale. =Scales in scale pocket relativeto “poison” glands, mucous glands, and annuli. b Margin and central part of scale, showing cells lining scale and the arrangementof the basal plate and the squamulae. on L. ZYLBERBERG AND M.H. WAKE GYMNOPHIONE SCALE MORPHOLOGY 31 blast (Fig. 21). The globules subsequently like structure. The collagen fibrils of the basal aggregate to form larger concretions. plate mineralize only within the squamulae. The collagenous stroma of the basal plate is The basal plate synthesized by the cells that line the basal surface of the scale. These flattened cells have a The basal plate is the most extensive part of voluminous central nucleus and form a continu- the scale, and is made up of superimposed plies ous sheet that is considered a pseudoepithelium of collagen fibrils. A diameter of -100 nm is composed of scleroblasts (Figs. 26,27). The rough achieved by most of the newly synthesized colla- endoplasmic reticulum (RER) is composed of gen fibrils that lie in the vicinity of the plasma short saccules. Mitochondria are not abundant membrane (Figs. 28,29,30). The collagen fibrils and the Gobareas are not well developed (Fig. of the basal plate are distinctly thicker than 13). Microfilaments are abundant; in some cells, those of the dermis (i.e., 100 vs. 30-nmdia; Figs. they appear to be aligned with microtubules and 35,36).In each ply, the collagen fibrils are packed with the newly synthesized collagen fibrils (Figs. in thick bundles, oriented in parallel (Figs. 13, 28,29), whereas in other cells such alignment is 26), and often, the periodic structure of groups of not observed (Fig. 30). The scleroblasts have fibrils is in register (Fig. 34). The direction of the long cytoplasmic processes that insert among fibrils varies from one ply to another; the angle of the collagen fibrils, thereby separating the fibrils rotation between two adjacent plies is about 90’. into distinct bundles (Figs. 13, 27). Collagen Thus, the plies constitute an orthogonalplywood- fibrils arise from these processes perpendicularly to the collagenous plies (Fig. 13). When a new ply is formed, the orientation of the first collagen fibrils synthesized is at a right angle to that of the preceding ply (Fig. 31); then an isolated Fig. 2. Longitudinal section perpendicular to the surface bundle is formed (Fig. 32), and finally the whole of the skin at the level of an annulus in Dermophis mexicanus newly synthesizedply is formed by collagen fibrils (light micrograph [LM]).The pouch(p) containsfouroverlap- ping scales (8) and lies below the epidermis (e). The scale aligned in the new direction (Fig. 33). pouch and the glands (mg = mucous gland, pg = “poison” gland) lie within the loose dermis (d) and do not penetrate the DISCUSSION dense dermis. Comparative morphology of dermal ossifications Fig. 3. Enlargement of outlined area of Figure 2 showing the outer part of the pouch (p) with the four scales (8). The The gymnophione condition mucou glands (mg)are located in the dermis (d) between the epidermis (e) and the scale pouch. A thin layer of connective The presence of dermal scales is unique to tissue (ct)separates the scales in their pocket. The squamulae gymnophiones among extant tetrapods. Various (sq) lie on the dorsal surface of the scales. workers (Taylor, ’72; Zylberberg et al., ’80; Per- Fig. 4. Longitudinal section (LM) of the inner part of a ret, ’82) have described the variation in scale pouch containing a folded scale (8) in Dermuphis mexicanus. morphology (e.g., size, shape, depth of base plate, pg = poison gland. distribution and shape of squamulae) among Fig. 5. Enlargement of outlined area in Figure 4. The two caecilian taxa. Certain features of scalation,such scales (s) are inserted in their own pocket (sp), surrounded by as overall distribution and presence or absence, connective tissue (d).The thick central part of the scale has have been used as reliable systematic characters. six plies, whereas the thin, folded part has only one ply. The We describe microscopic features of scale mor- squamulae (sq)top the basal plate. The margin of the scale (solid arrow) is not lined by scleroblasts. phology that might be taxonomically useful. Among adults, the shapes of the squamulae on Fig. 6. The thin layer of connective tissue (ct) between the scales and the pattern of deposition of miner- two scales separates the pockets (sp); transmission electron alized material seem to be fairly consistent. Such micrograph (TEM); longitudinel section (Ls).Fibroblasts (IC) line the pocket. The scale is surrounded by the sclero- features are best examined by scanning electron blasts (sc).Dermophis mexicanus. bp = basal plate, sq = microscopy, so that details of structure can be squamula. assessed large samples of scales have yet to be evaluated for any single taxon. A comparison of Fig. 7. Detail of the dorsal part of a pocket limed by a fibroblast (IC) in Microcaeciliu unicolor (TEM LS). The the scanning electron micrographs of Hypogeo- scleroblast (sc) lies on the squamula (sq). p = pouch; sp = phis (Casey and Lawson, ’79), Ichthyophis and scale pocket. Hypogeophis (Zylberberg et al., ’80), Geotry- petes and Herpele (Perret, ’82), Dermophis Fig. 8. Detail of the basal part of a pocket in Dermophis mexicanus (TEM, LS). The scale pocket (sp) is lined by a (Wake and Nygren, ’87), and those in Taylor’s fibroblast (IC).bp = basal plate; p = pouch; sc = scleroblast. atlas (’72) reveals considerable variation in squa- 32 L. ZYLBERBERG AND M.H. WAKE

Figures 9-12 GYMNOPHIONE SCALE MORPHOLOGY 33 mular shape and structure. The rectangular blasts that line the scale pocket of gymno- shape of peripheral squamulae in Ichthyophis phiones do not differentiate as in fishes (see (Zylberberg et al., ’80) contrasts markedly with Whitear et al., ’80). In osteichthyans, the outer the rounded shapes of Geotrypetes (Perret, ’82) surface of the elasmoid scale is connected to the and Dermophis (Wake and Nygren, ’87). It superficial dermis by collagenous anchoring bun- should be noted that these contrasts are greatest dles that arise from the outer surface of the scale among peripheral squamulae;examination of the and extend through the dermis to the epidermal- published photomicrographs indicates that the dermal junction (Zylberberg and Meunier, ’81; centers of scales bear squamulae that are both Sire, ’85).Such bundles are absent in the gymno- rounder and more irregular in shape. A common phione scales examined. This phenomenon may feature of squamulaeof virtually all species exam- be related to the location of the scale rather than ined is that mineralized material is deposited in to the structure of the squamulae. In gymno- a globular manner (see photomicrographs cited phiones, the scales are well inserted within the above). The only exception to this pattern that dermis among skin glands, and they are covered we have observed is in Caecilia tentaculata, in by a thick pleuristratifiedepidermis (Gabe, ’71a). which deposition is essentially smooth (Wake Similarly, the reduced scales of the eel which are and Zylberberg, unpublished data), and we are covered by a mosaic of smooth squamulae do not examining this situation further. have anchoring bundles. These scales also are Comparison with osteichthyan scales covered by a thick pleuristratified epidermis (Zyl- berberg et al., ’84). In further contrast, the large The pouch and the pocket scales of Protopterus, which are located in a very Our data confirm that gymnophione scales loose dermis close to the thin epidermis, have have peculiarities apparently related to their lo- squamulae with well-developed collagenous an- cation in the segmentally arranged annuli, and choring bundles (Zylberberg,’88). that they share more structural similarities with the scales of extant osteichthyans than with the Squamulae osteoderms of extant amphibians and reptiles. Our observations confirm that mineralization Because of their position relative to the annuli, in gymnophione scales is limited to the squamu- the overlapping scales in gymnophiones occur in lae, which are isolated plates topping the scales; narrow transverse rows that partly or completely mineralization does not occur in the basal plate encircle the body. In osteichthyans, the imbri- as described by Casey and Lawson (’79). Gymno- cate scales are distributed over the body com- phione squamulae have two simultaneous pro- pletely or in patches, without an obvious segmen- cesses of mineralization, termed spheritic and tal association.Our data indicate that every scale inotropic (Orvig, ’68). These two processes are in both species examined lies within its own the result of the heterogeneous composition and pocket, as do the elasmoid scales of osteichthy- organization of the extracellular matrix in the ans (Whitear et al., ’80). Recent descriptions, squamulae. Recent reports point out the impor- including those at the ultrastructural level, have tance of the organic matrix in controlling crystal mentioned that several overlapping scales lie in a deposition (reviewed by Weiner, ’86). Where “pocket” (Taylor, ’72; Gabe, ’71a; Wake, ’75; spheritic mineralization occurs, the shape of the Zylberberg et al., ’80; Fox, ’83). However, as spicules depends on the radiating arrangement noted above, that “pocket” is actually a large of the noncollagenous matrix. The crystals asso- connective tissue pouch associated with an annu- ciated with the collagen fibrils are aligned along lus. The pouch contains the scales, each of which the fibrils so that their long axis (the crystallo- lies in a thin, connective tissue pocket, homolo- graphic c axis) is parallel to the collagen fibril gous to that of osteichthyans. However, the fibro- axis (Schmidt, ’36; Stuhler, ’38). Moreover, the crystals are distributed according to the axial periodicity of the collagen fibrils as first described in bone (Hodge and Petruska, ’63; Glim- Fig. 9. Randomly selected scale of Microcaecilia unicolor cher and Krane, ’68; Glimcher, ’81; Arsenault, stained with kin-redto show shape and arrangement of ’88). squamulae. According to Orvig (’68),spheritic mineraliza- Fig. 10. Randomly selected scale of Dermophis mexica- tion may be considered the phylogenetic precur- nus stained with alizarin-red. sor of inotropic mineralization. He considered the latter to represent the “ultimate stages” in a Fig. 11. Scanning electron micrograph (SEM) of aquamu- lae of Microcaecilia unicolor. phyletic process of increasing complexity of “calcification mechanisms.” Spheritic mineraliza- Fig. 12. Squamulae of Dermophis mexicam (SEM). tion also may be considered an ontogenetic pre- Figures 13-17 GYMNOPHIONE SCALE MORPHOLOGY 35 cursor of inotropic mineralization,because during ticipate in the mineralization processes have not dentine ontogenesis, spheritic mineralization been identified. (which forms globular dentine) is replaced by Basal plate inotropic mineralization (Keil, '39; Orvig, '67; Poole, '67). Therefore, the squamulae of gymno- In gymnophiones,the thick collagenous fibrils phiones, the outer surface of osteichthyan scales forming the basal plate are organized as a ply- (Sire, '85; Zylberberg, '88 and unpublished data), wood-like structure, but it is less regularly ar- and the osteoderms of reptiles (Levrat-Calviac ranged than in the elasmoid scales of teleosts and Zylberberg, '86) have retained a primitive and sarcopterygians. The reduction of organiza- type of mineralization that occurs concomitantly tion of the basal plate in gymnophiones may be with a "more advanced" mineralization process. associated with a generalized reduction of the The mineralized spherules are more abundant dermal skeleton in both fishes and tetrapods. on the outer surface of the squamulae, but also The most reduced osteichthyan scale observed are found on the inner surface among the colla- occurs in the eel in which the basal plate has only gen fibrils. These spherules, which have a radia- one ply (Zylberberg et al., '84). The scales of tion arrangement of crystals, do not correspond gymnophiones are thin and the plies are much to the Mandl's corpuscles characteristic of elas- less numerous than in fishes. However, the basal moid scales. In Mandl's corpuscles, the crystals plate, which shows the usual histological and are oriented by the alignment of the collagen histochemical characteristics of lamellar bone, fibrils (Schonborner et al., '81). The limit be- does not mineralize as it does in the basal plate tween the squamulae and the basal plate is rep- of osteichthyans (Meunier, '84). According to resented by the mineralizing front, though colla- Meunier, thick basal plates made of isopedine gen fibrils arise from the basal plate and that has lost its ability to be mineralized reflect a penetrate the squamulae. These fibrils are not general trend toward reduction of the dermal part of the plywood-like structure of the basal skeleton. plate itself. As noted by Zylberberg et al., ('80), The collagen fibrils of gymnophione scales, the TEM micrographs of well-developed scales like those of fish, are thicker than those of the show mineral deposits both along the collagen surroundingdermis, including the collagen fibrils fibrils and within the interfibrillary matrix, as that separate the bundles forming the plywood- well as in spherules,where they are arranged in a like structure of the base plate and those that radiating pattern. Therefore, the site of nucle- cross the scale perpendicularly to the plies. The ation cannot be determined (present study; Zyl- latter collagen fibrils differ from the TC fibers berberg et al., '80). Moreover, the cells that par- described in the elasmoid scales of Cyprinidae. They are thinner than the collagen fibrils of the plywood-like structure (Onozato and Watabe, '79) and are involved in the first stages of miner- Fg.13. Scale of Microcaecih unicolor sectioned perpen- alization of the basal plate (Zylberbergand Nico- dicular to its surface (TEM). The fibroblasts (IC) forming the las, '82). As in fish scales, the collagenous stroma scale pocket surround the scleroblasb (sc). The outer part of as well as the noncollagenous extracellular ma- the squamula (sq)has abundant mineralized globules. In the inner part of the squamula the minerahng front (mf) is trix are synthesized by the cells surrounding the located in the basal plate (bp). The basal scleroblasts have scales, which also control mineral deposition. long processes (arrows) that separate the bundles of collagen Therefore, the gymnophione scale is an acellular fibrils. tissue as defined by Meunier ('87); further, the Fig. 14. The inner part of the squamulae (sq) is PAS- basal plate may be considered a bony tissue reactive (dark color) whereas the outer part shows weak which has lost its capacity to be mineralized staining with alcian blue (pH 2.5) (arrows) (LM, LS). The (Meunier, '84). basal plate does not react to either stain. Dermophis mexica- The scleroblasts involved in the formation of nus. the scales line each scale. They are contiguous Fig. 15. The inner part of the squamulae (sq)is strongly only along the inner surface of the scale where reactive to tetrazolium, though the outer part (arrows) and they form a pseudoepithelium. Because they syn- the base plate (bp) stain weakly (LM LS). Dermophk mexi- thesize the thick collagen fibrils that form the canus. basal plate, they are assumed to control fibrillo- Fig. 16. The outer surface of the squamula (sq) has many genesis and the orientation of the collagen as has mineralized globules (TEM LS). Dermophis mexicam. been described for fish scales (see above). The long cytoplasmicprocesses that penetrate deeply Fig. 17. Demineralized (EDTA)scale section fromDermo- phis mexicaw (TEM LS). Theglobulea are primaily at the within the basal plate are involved in the forma- outer part the squamula They do not show any organic tion of the bundles that form the superimposed matrix. ct = connective tissue;mf = mineralizing front. plies. However, the processes seem to orient the Figures 18-24 GYMNOPHIONE SCALE MORPHOLOGY 37 collagen fibrils that arise from the cell processes scale. A similar rim of marginal cells is absent in perpendicularly to the plies. An obvious coinci- gymnophiones. dence of the orientation of cytoskeletal elements Comparison with dermal ossifications in other (superficial microtubules and actin filaments) amphibians with the innermost collagen fibrils has been described (Zylberberget al., ’%), and indicates that Some anurans and salamanders have dermal cytoskeletal organization also is involved in the ossifications, but not scales. Members of four orientation of collagen fibrils. Such relationships families of frogs independently have evolved os- between the cytoskeleton and newly synthesized teoderms-bony plates that occur in the dermis collagen fibrils are not observed in the basal on the dorsum of the body, and in some on the scleroblasts of gymnophione scales. Therefore head and the limbs. Ruibal and Shoemaker (’84) the less well organized plywood-like structure of carefully examined the structure of frog osteo- the basal plate in gymnophione scales may be derms, and reported considerable variation related to reduced involvement of the cytoskele- among families, including vascularized bony ton in the orientation of fibrils produced by the plates that bears spines that protrude into the scleroblasts. The scleroblasts are connected to epidermis and avascular osteoderms composed each other and are not able to modify their of calcified collagen fibrils in a three-dimen- shapes, as do isolated fibroblasts (Birk and Trel- sional arrangement. Mineralization is appar- stad, ’84; Birk and Trelstad, ’86). ently inotropic. Most of the cells surrounding well-developed Members of all three orders of amphibians gymnophione scales do not appear to be actively possess another pattern of dermal ossification, secretory, suggesting that increase in size and in co-ossification of the skin of the head to the thickness of developed scales is slow, if it occurs skull. Trueb (’66)described co-ossification in the at all. In teleosts, increase of thickness of devel- skull of the casque-headed frog, Hyla septentri- oped elasmoid scales is primarily a contribution onulis. Extensive bony protuberances of skull of basal scleroblasts, which have a low rate of bones and of the dermis bind skull, dermis, and synthesis (Waterman, ’70; Frietsche and Bailey, epidermis. In the plethodontid salamander Anei- ’80). Increase in size results from the activity of des lugubris, spikes of dermal bone protrude marginal scleroblasts that form a rim around the into the epidermis and there is virtually no loose dermis (D. B. Wake, personal communication). In gymnophiones, much of the dermis on the dorsal part of the skull and the margins of the jaws has been eliminated, and dermal fibers that Fig. 18. Partially demineralized section from Microcae- overlie the skull and bind it to the epidermis cilia unicolor (TEM, LS). The globules have concentric spheres of mineralization. have been mineralized (Wake,unpublished data). Several taxa of extinct amphibians among the Fig. 19. Scale of Microcaecilia unicolor treated with Labyrinthodontia, Aistopoda, and Microsauria EDTA and ruthenium red (TEM). The globules are lined by possess scales. Colbert (’55) and Olson (’79)exam- an electron-dense material (arrows).Some also have an electron-dense central core. ined patterns of scalation in members of these groups, particularly the labyrinthodont Trimer- Figs. 2&22. Formation of globules of mineralization in orhachis. The scales of Trimerorhachis are thin Microcaecilia unicolor (TEM). and somewhat elongate, less round than in other Carboniferous scaled amphibians. The scales Fig. 20. The crystals (arrows)appear in globules near the vicinity of a scleroblast (sc). have a basal layer with a pattern of concentric rings, assumed to be growth rings by Colbert, Fig. 21. The crystals are arranged in a radiating pattern. and a superficial layer of longitudinal ridges, The globules lie near the scleroblast (sc). Small globules are giving a “corrugated” appearance to the surface. agprwtinp. Both layers are well mineralized. Olson, how- Fig. 22. The globule has the same location and the same ever, considered these structures to be layers of organizationas it increases in size. sc = scleroblast. osteoderms having no resemblance to cycloid Fig. 23. Mineralizing front (mf) at the inner part of a scales. There appear to us to be both terminolog- squamula (sq) (TEM LS). Mineralized globules lie among ical and analytical problems with these interpre- the collagen fibrils of the basal plate (bp).Dermophis mexica- tations, Significantly, both workers suggested nus. that trends in evolution of dermal structures were similar and parallel in amphibians and tele- Fig. 24. Periphery of a squamula (sq)of Dermophis mexicanus (TEM, LS). Isolated globules are abundant (arrows) osts, with reduction of size, composition, num- among the collagen fibrils of the base plate (bp). bers, and distribution of scales. Colbert sug- ,- Figures 25-30 GYMNOPHIONE SCALE MORPHOLOGY 39 gested that the evolutionary patterns represent tangled microfilaments that occur among the convergences between amphibians, descended collagen bundles. Both inotropic and spheritic from crossopterygians with rhomboid ganoid mineralization take place in these osteoderms. scales, and teleosts, descended from palaeonisc- The osteoderms are strikingly continuous with ids that also had rhomboid ganoid scales. Be- the deep dermis, and are joined to each other by cause there is virtually no evidence from which dense bundles of collagen fibrils. Osteoderms patterns of mineralization can be deduced, this grow concentrically, and exhibit electron-dense debate cannot be resolved. growth rings, such as occur in the bones of all vertebrates (Castanet, ’81). Comparison with dermal ossifications of Levrat-Calviac and Zylberberg (’86) consid- reptiles ered osteoderms to be secondary formations. Osteoderms, or mineralized dermal plates lo- These data and conclusions largely corroborate cated in the skin, occur in many squamate rep- those of Zylberberg and Castanet (’85) for the tiles. Levrat-Calviac and Zylberberg (’86) re- osteoderms of Anguis fragilis, though osteo- viewed the literature on such structures as part derms of the latter differ from those of T. mauri- of their careful assessment of the histology and tunica in being small, flat, disc-shaped, and asso- cytology of osteoderms in Tarantola mauritan- ciated with the epidermal fold of the overlying ica. They noted much variation in location,devel- epidermal scale. It is significant that osteoderm opment, and correlation with epidermal scales, formation occurs without differentiation of a relationship with adjacent tissues, and general specialized tissue structure, such as the dermal structure, though there is consistencywithin spe- papilla of the fish scale. It is likely that these cies. Their analysis revealed that osteoderms of observations characterize osteoderm formation T.mauritanica have two components. A miner- and structure in many squamates, with lineage- alized basal layer is composed of many closely specific variation in location, size, and shape. packed collagen fibrils effectively continuous with Evolution of dermal ossifications those of the dermis. An outer layer, in the superficial loose dermis, is crossed by rather few colla- Dermal scales in gymnophiones and osteich- gen fibrils that arise from the basal layer. These thyans, and osteoderms in frogs and squamate fibrils connect the osteoderm to the loose der- reptiles, share a number of common properties. mis. The outer, superficial, layer contains miner- For example, squamate osteoderms bear a strong alized globules that surround the mineralized superficialresemblance to the osteoderms of frogs collagen bundles. Within the globules, crystals of as described by Ruibal and Shoemaker (’84) in mineralization are deposited on a matrix of mi- their dermal continuity, variable location, and crofibrils that is composed of radially oriented, apparent mineralization pattern, though they lack the dorsal spikes exhibited by many frog osteoderms. Further, the general structure with a distinct basal layer and a differently mineral- Fig. 25. Margin of the scale in Dermophis mexicanus ized superficial or outer layer is similar among (TEM).The scale (a) is in contact with the connective tissue osteoderms to the dermal scales of osteichthyans (ct)of the scale pocket. and of gymnophiones. The similarity is en- Fig. 26. Basal scleroblasts (sc) of Dermophis mexicanus hanced by evidence that mineralization in all synthesizing the thick collagen fibrils of the basal plate (bp) these groups usually is both inotropic and (TEM). The collagen fibrils oriented in parallel are not packed spheritic; this suggests common properties of in bundles. association with collagen fibrils and their sur- Fig. 27. Basal scleroblasts (sc) have long processes (ar- rounding matrix. Moss (’72) commented on the rows) which separate bundles of collagen fibrils (TEM). Der- series of homologous developmental events in- mophis mexicanus. volved in the formation of diverse dermal struc- Fig. 28. Enlargement of outlined area of Figure 27. The tures (e.g., teeth), and emphasized the common collagen fibrils and cytoskeletal components (microtubules properties of collagen and “ground substance.” [mow], microfilaments [double mows]) show the same ori- He presented a classification of integumental entation. The collagen fibrils are in contact with the sclero- skeletal structures. Meunier and Geraudie (’80) blast. presented cogent arguments about the common Fig. 29. Some microfilaments (arrows) have the same pattern of development of dermal ossification, orientation as the collagen fibrils (arrowheads) in contact with particular reference to scales. They de- with the cell (TEM). Microcaecilia unicolor. scribed the spatial organization of collagen as a plywood structure in the both basement lamella Fig. 30. No relationship of the direction of the cytoskeleton (arrows) and of the collagen fibrils (asterisks) is discem- and the dense dermis, including the basal layer able (TEM). Microcaecilia unicolor. of teleost scales. The scales have thicker fibrils ,,. L. ZYLBERBERG AND M.H. WAKE

Figures 3136 GYMNOPHIONE SCALE MORPHOLOGY 41 than in the associated dermis (we describe the shared in many ways with osteoderms). We par- same phenomena in gymnophiones). They con- ticularly object to the notion of common ances- cluded that the contre-plaque structure is a com- try. Earlier workers did not so misconstrue; even mon property of dermis. Different taxa have Colbert (’55),although associatinggymnophione different “aptitudes” for mineralization, depen- scales with those of possible extinct amphibian dent upon the macromolecular arrangement of ancestors, called teleostean and gymnophione the collagen fibrils and the biochemical environ- scales a convergence among “active” vertebrates. ment in which they occur. Kerr (’55) found a striking “convergent simi- The data available on the structure of gymno- larity” between the scales of Neoceratodus and phione and osteichthyan scales and frog and Amia, but the resemblance of the scales of the squamate osteoderms support the hypothesis other lungfish to teleost scales to be more super- that such mineralized dermal structures occur ficial. He concluded that lungfish scales have because of the structural properties of the der- only a general resemblance to gymnophione mis, common to all vertebrates. Expressions of scales. It is clear that attributing common ances- these properties arise in a number of lineages, try to dermal scales of osteichthyans and gymno- and have a diversity of manifestations. We con- phiones rests on little or no significant evidence. sider it inappropriate to speculate about the It is equally difficult to associate gymno- “selectiveforce(s)” that might have favored such phione scales with those of extinct amphibian new structures. Instead, we offer a structuralist, groups proposed as ancestral. Various groups a-historicalexplanation for the similarity of struc- have been suggested, but there is a paucity of ture of the dermal ossifications and their appar- evidence. Further, there is not yet an agreed- ent modes of mineralization, despite the signifi- upon phylogeny for all amphibians. For exam- cant differences in size, shape, and location of ple, Carroll and Currie (’75) considered micro- such structures. saws to be ancestors of gymnophiones;Gardiner We are concerned that an assumption of ho- (’83), based only on vertebral structure, consid- mology of the dermal scales of osteichthyans and ered nectridians to be the sister group of extant gymnophiones as a consequence of descent from a hypothetical ancestral dermal scale has crept amphibians, and microsam to be amniotes! Be- into the literature (see, e.g., Ruibal and Shoe- cause there are significant differences in struc- maker, ‘84, p. 324). We believe that assumptions ture of scales of extinct amphibians and of gym- of homology of osteichthyan and gymnophione nophiones, and there is no soft tissue information scales are based only on (1)their dermal origin, available for the former, we consider it equally and (2) their similarities of shape and some as- plausible that scales might have arisen indepen- pects of structure (despite the fact that these are dently in several lineages of amphibians, including gymnophiones. Clearly dermal ossifications have arisen numerous times; differences in size, shape, location, pocket structure, etc., of amphib- Figs. 31-33. Formation of a new ply in the basal plate in ian scales and the mineralized dermal structures Dermophis mexicanus (TEM LS). of other vertebrates suggest lineage-specificvari- ation and, probably, origin, but from a tissue of Fig. 31. The collagen fibrils (mow) in contact with the scleroblast are directed approximately perpendicular to the origin that has common structural capabilities previously deposited layer of fibrils. bp = basal plate. (and constraints).Furthermore, the unique structural properties of gymnophione scales (particu- Fig. 32. A bundle of collagen fibrils (arrow) in contact larly the segmental association and other aspects with the scleroblast perpendicular to those. forming the ply beneath. of location and structure) suggest a de nom acquisition in the lineage. Fig. 33. Collagen fibrils in contact with the scleroblast are We agree with Ruibal and Shoemaker (’84) perpendicular to those of the adjacent ply. that the term “dermal scale” should be used for Fig. 34. Longitudinal section of collagen fibrils of a scale the mineralized dermal units of both osteichthy- of Dermophis mexicanus (‘EM). The peridcities of both ans and gymnophiones,and “osteoderm”for the fibrils are in register. dermal structures of frogs and squamates. This terminology recognizes certain convergent at- Fig, 35. Cross section of the collagen fibrils of the basal plate of a Dermophis mexicanus scale (“EM).sc = sclero- tributes of shape and structure (scales being blast. structures that arise from differentiating dermal fibroblasts,lie in pockets, and are generally spher- Fig. 36. Cross section of the dlagen fibrils forming the plywood ofthedensedermis inllermophismexicanwl (”EM). oid; osteoderms being metaplastic structures oc- Note the difference in diameter of hesal plate (Fig. 35) and curring in preexisting dermal tissue and strongly densedermis collagen fibrils. connected to the dense dermis), but it does not 42 L. ZYLBERBERG AND M.H. WAKE carry any connotation of common ancestry or of Fox, H. (1983) The skin of Ichthyophis (Amphibia: Caecilia): developmental process. We conclude that, at An ultrastructural study. J. Zool. Lond. 199:223-248. Frietsche, RA., and C.F. Bailey (1980) The histology and this time, the dermal ossifications of vertebrates calcification of regeneratingscales in the blacksspotted top- appropriately are regarded as diverse expres- minnow, Fundulus oliuaceus (Storer).J. Fish. Biol. 16.692- sions of the common structural propensity for 700. mineralization of the dermis itself. Gabe, M. (1968) Techniques histologiques. Masson et Cie, Paris. ACKNOWLEDGMENTS Gabe, M. (1971a) Don6es histokgiques sure le tegument d’Ichthyophis glutinosus L. (Batracien, Gymnophione). We thank Francoise Allizard, Enrique Lessa, Ann. Sci. Nat., ZooL, Paris, 12erne series 13:573-608. Wendy Marlor, and Kristen Nygren for techni- Gabe, M. (1971b) Apport de I’histologie a l’etudedes relations cal assistance, and Jean Lescure, Theodore Pap- phylBtiques des Gymnophiones. Bull. BioL 105t125-157. enfuss, Robert Seib, David Wake, and Thomas Gardiner, B.G. 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